The present invention relates to a novel container and process, and more specifically, to a rectilinear plastic container whose interior is divided into two or more channels which is filled with a microbial growth medium and is utilized in a process for enumeration of microorganisms found in clinical or industrial samples. Different microbial growth media may be placed in adjacent channels of the rectilinear plastic container for the process of the present invention. The process of the present invention demonstrates a more efficient and rapid means to identify and enumerate microorganisms in a sample.
Analysis of various specimen samples for the quantity of microorganisms they might contain is routinely done. Analysis is also performed to determine the identity of microorganisms in a sample. Samples can come from many sources and include clinical, i.e., urine, blood, pus, etc., and industrial (e.g. food, water, environmental or pharmaceutical) sources.
Enumeration and identification of the number of microorganisms, including yeasts, bacteria and fungi, in a sample is important for many reasons. This knowledge can assist a doctor in determining the level of and the type of infection in a patient and can therefore assist in determining appropriate treatment. Enumeration and identification of bacteria or other microbes from water and wastewater samples is routinely done to see if the water meets acceptable health standards. Likewise, food products such as meats and dairy products are also tested.
Methods of enumerating microorganisms are known, but suffer from significant drawbacks. In perhaps the most routinely used method, the sample suspected of containing microorganisms is serially diluted, typically in ten-fold dilutions but also in two-fold or five-fold dilutions. Making five or six serial ten-fold dilutions is typical. Each diluted sample is then spread onto a separate plated agar-based medium using a constant volume, for example 10, 50 or 100 uL of each dilution. To ensure accuracy, these samples are usually plated in duplicate or triplicate. The inoculated plates are then incubated. Incubation periods of 16-24 hours are standard. After the incubation period, the plates are examined for growth of microorganisms.
Typically, some plates in the dilution series will show little or no growth, while others will show growth of so many colonies that accurately counting them is not feasible. Typically, one or two plates at a specific dilution will yield a countable number of colonies. Generally, plates demonstrating between 30 and 300 colonies are considered countable and representative of the starting viable population. Knowing the dilution of the sample used on the counted plate, one can readily calculate the number of microorganisms in the original sample.
As the description of this process makes clear, this so-called colony enumeration method is both time consuming and, labor intensive and thus expensive. For each sample tested, many media-containing plates are prepared and used, while only a few yield results which provide useful information.
Thus, many plates are in effect wasted, significantly increasing the cost of conducting such a test. Moreover, waiting for the incubation of the microorganisms until there are visually observable colonies takes at least one working day. In some instances, for example where the public health is involved, such a waiting period can delay implementation of decisions needed to prevent further cases of diseases caused by the microorganism.
An alternative to this well accepted enumeration technique was suggested in Biotechniques, vol. 23, 648 (1997). The proposed alternative used a track-dilution technique whereby six 10-fold serial dilutions of a sample containing an unknown quantity of bacteria were plated onto a single square agar plate. 10 uL samples were spotted in a column on the agar surface along one side of the square using a micropipet in ascending order of bacterial concentration. The plate is tipped at an angle to allow the samples to run down the plate and then allowed to dry before incubation. This method used less materials and took less time since fewer plates were inoculated. The results achieved were not statistically different from the counts achieved with the separate plate method.
While this method is an improvement over the prior method, it still suffers from drawbacks. For example, the possibility that the different tracks of the dilution will cross over into each other exists when the plates are tilted. Further, this method requires the use of a single growth media in the plate. Additionally, the possibility of crossover makes such a system difficult to automate.
It is therefore an object of the present invention to have a device used to enumerate microorganisms where there is no possibility of crossover contamination between adjacent samples. It is also an object to have a device where identification and enumeration can be simultaneously performed.
It is also an object of this invention to permit greater flexibility in the testing and enumeration process by permitting the enumeration or identification of more than one microorganism in a sample.
Another object of the invention is to decrease the time to analyze and enumerate outgrowth of organisms in samples.
Another object of the invention is to determine susceptibility of microorganisms to antibiotics by establishing concentration gradients, in an appropriate gelled growth matrix, within each channel.
Another object of the invention is to allow portions of the same sample to be inoculated onto different media.
Automation of microbial enumeration is yet another object of the present invention.
The present invention is a square or rectangular plastic dish, also referred to as a rectilinear plastic container having a depth sufficient to contain a microbial growth medium in an amount that is adequate to support growth of microorganisms. The dish preferably has a lugged lid which can preserve the moisture level of the growth media as well as its sterility and the chemical integrity of the growth media. This dish is preferably separated by a predetermined number of spacers to create parallel channels or spaces which run the length of the dish. The troughs thus formed may be of uniform depth or may be formed so as to create a wedge-shaped profile.
Microbial enumeration is performed by placing a sample that is known or suspected of containing a microorganism, at a predetermined dilution, at the top of each channel. The dish is then tilted to allow the sample to flow along the length of the channel. Alternatively, a spreading device is used to distribute the sample from one end of a channel to the other. Appropriate incubation conditions are applied. After a sufficient amount of time has passed to permit microbial growth the channels are inspected either mechanically, by a high-resolution camera, for example, or visually. Based on the microorganism counts made at this time, the total number and concentration of microorganisms in the original sample can be calculated.